European Journal of Forensic Sciences

Original Research
www.ejfs.co.uk
DOI: 10.5455/ejfs.203527

Method validation for simultaneous

determination of pesticide residues
in post-mortem samples by high-
performance liquid chromatography-
ultraviolet method
Deepti S Deshpande, Ashwini K Srivastava

Department of Chemistry,
University of Mumbai,
Vidyanagari, Santacruz
(East), Mumbai 400098,
Maharashtra, India
Address for correspondence:
A. K. Srivastava,
Department of Chemistry,
Lokmanya Tilak Bhavan,
University of Mumbai,
Vidyanagari, Santacruz
(East), Mumbai - 400 098,
Maharashtra, India.
E-mail: aksrivastava@chem.
mu.ac.in.
Received: October 02, 2015
Accepted: February 08, 2016
Published: 26 February, 2016
ABSTRACT
Introduction: In forensic or clinical toxicology, a correct interpretation of toxicological findings needs reliable
analytical data. The analytical methods that are to be used for routine analysis need careful method development
and thorough validation. Blood, body fluids, and certain organs are mostly used in examinations of deaths due to
intoxication. However, validated methods for the analysis of post-mortem samples, especially tissues are very
few. The aim of this work was to validate a simple reversed-phase high-performance liquid chromatography-
ultraviolet method for the simultaneous determination of carbamate (propoxur), organophosphorus (malathion,
quinalfos, profenofos, and chlorpyrifos), and organochlorine (endosulfan) pesticides in post-mortem tissue and
blood samples. Materials and Methods: The pesticides incorporated in the method were those commonly
encountered in suicidal or homicidal poisonings in India. Before analysis, QuEChERS isolation technique was
conveniently used to clean the tissue and blood samples. Validation proved the applicability of matrix-matched
calibration for routine screening of the pesticides in post-mortem samples. Results: The working range of all
the pesticides showed good linearity with correlation coefficients (r) ranging from 0.9996 to 0.9999. The limits
of detection for all pesticides range from 0.37 to 0.70 μg/g in tissue while it varies from 0.14 to 0.35 μg/ml in
blood. The limit of quantitation ranges from 1.16 to 2.50 μg/g in tissue and from 0.44 to 1.50 μg/ml in blood.
Conclusion: The developed method can be effectively used for routine screening and quantitation of the
pesticides belonging to diverse groups which otherwise require different time consuming or costly analytical
techniques for confirmation and quantitation "
KEY WORDS: Forensic sciences, forensic toxicology, liquid chromatography, method validation, pesticides

INTRODUCTION the lower limit of quantification. At the same time, they

The process of method validation has a direct impact on
the quality of residue analytical data. Forensic laboratories
are specifically in need of effective methods for the correct
interpretation of toxicological findings. Unreliable data can
lead to unjustified legal consequences or to wrong treatment
of the patient. The importance of method validation is
emphasized by Peters et al. [1] not only to develop a method
carefully for the correct interpretation of the toxicological
findings in daily routine work but also to demonstrate
the inherent quality of any analytical method to prove its
applicability for a certain purpose, especially in the context
of quality management and accreditation. As mentioned by
them as a general agreement for quantitative bio-analytical
procedures, the essential validation parameters to be evaluated
are selectivity, calibration model (linearity), stability, accuracy
(bias), precision (repeatability, intermediate precision), and
also mention that though no general validation guideline is
available for a qualitative procedure, there is an agreement
that at least selectivity and limits of detection (LOD) are
significant evaluation parameters with additional parameters
such as precision, recovery, and robustness. Although there are
no set maximum limits for the presence of pesticides in post-
mortem tissues in clinical and forensic toxicology, a precise
analytical method is favored for procuring authentic results
to prove the intake of a substance.
Cases involving fatalities due to consumption of pesticides
are common in India. A study of suicidal behavior in India
indicates that poisoning (93%) is the most common mode
of deliberate self-harm irrespective of gender [2]. It also
reveals that pesticides are the most common lethal poisons
in the household as they are easily available and due to
the ignorance and carelessness about their self-storage [3].

Eur J Forensic Sci ● Jul-Sep 2016 ● Vol 3 ● Issue 3 1

Deshpande and Srivastava: Pesticide residues in post-mortem samples
Among the pesticides, the organophosphorus group (OP) is
responsible for the largest number of deaths followed by the
organochlorines (OCs) and carbamates (Carbs). The OPs and
Carbs are cholinesterase inhibitors while OCs impair nervous
system function by depolarization of the membrane. When
post-mortem tissue or blood in such poisoning cases is referred
to clinical/forensic toxicology, the correct interpretation
of toxicological findings is an important objective of the
toxicologist for which a reliable analytical method becomes
an important prerequisite.
Literature survey shows documentation of various reviews
on analytical methods for monitoring pesticides and their
metabolites in different matrices such as rice, fatty vegetables
and food, fruits, and vegetables [4-14]. An increased trend
of gas chromatography and liquid chromatography with MS
in the analysis of pesticides [15-19] is evident from a large
number of reviews. High-performance liquid chromatography-
ultraviolet (HPLC-UV)-visible detection is common for
analysis of pesticides belonging to one particular class such
as the OPs, OCs, or Carbs [20-27], wherein the pesticides
are determined individually or with other pesticides of the
same class. Blood, urine, or plasma is the most common
matrix of determination in most of the reported analytical
methods [28-36] while very few methods document their
analysis in bone marrow [37], hair [38], or tissue materials
[39,40]. A recent work reports HPLC with photodiode array
detection for pesticide analysis in bovine tissue [41]. Similarly,
QuEChERS method of sample preparation since its inception
by Anastassiades et al. [42], has not only found its application
for matrices such as foods [43], fruits, and vegetables [44] but
also for matrices such as sea-food, beef, and human whole
blood [45]. However, it is obvious from one of the latest
reviews that there is a lack of method developments in post-
mortem tissues and blood though there is an array of analytical
methods to determine pesticides in several matrices.
Method validation is problematic in post-mortem analysis
because not only the acquisition of representative validation
data virtually impossible owing to varying composition of
samples but also the concentration of the analyte is no longer
what it was at the time of death. The composition of partly
decomposed or putrefied samples may vary considerably from
case to case, and hence, validation studies carried out using
matrix samples from one or several post-mortem cases may
become questionable. However, if the tissues are properly
preserved and are not decomposed before analysis, then the
validation data can ensure sufficient quality of the results.
Validation studies are certainly justified if an analytical
method is to be used routinely. Since in India deaths
due to pesticide consumption is a common phenomenon
the analysis of multiple - class pesticides in post-mortem
tissues and blood is a routine. Keeping in view the above
considerations, the simple reversed-phase HPLC-UV method
is validated, for the simultaneous determination of pesticides
commonly found in poisoning, viz., propoxur (Carbs),
malathion, quinalfos, profenofos, chlorpyrifos (OPs), and
endosulfan (OC).
2 Eur J Forensic Sci
MATERIALS AND METHODS
Chemicals and Reagents
All reagents were of HPLC grade (unless otherwise specified)
and used as supplied by the manufacturers. High purity
water, acetic acid, and acetonitrile (ACN) were procured from
Merck Specialties Pvt. Ltd., India. The pesticide standards
(Malathion, Quinalfos, Profenofos, and Chlorpyrifos) were
from Sigma-Aldrich, India. Propoxur and endosulfan were
from Accustandard, India with a certified purity of ≥97%.
The Agilent SampliQ QuEChERS kits procured from Agilent
Technologies, India were made up of an extraction kit and a
dispersive SPE kit.
SampliQ QuEChERS extraction kit: The kit consists of 50 ml
extraction tube and pack of 6 g magnesium sulfate and 1.5 g
sodium acetate.
SampliQ QuEChERS dispersive solid phase extraction (SPE)
kit: The kit consists of 15 ml and 2 ml SPE tubes containing
primary secondary amine (PSA), C18 and magnesium sulfate
and was used for clean-up of extracts obtained after extraction
with ACN.
Materials
The post-mortem tissue sample and blood were obtained from
the Forensic Science Laboratory, Mumbai. The samples used
for development and validation of the method were those
which were confirmed to be negative for any toxicological
findings after routine analysis while the samples used to
demonstrate the applicability of the developed method were
those which had poisoning history and were confirmed positive
after the routine analysis. The samples were stored at −4°C
until analysis.
Preparation of Different Solutions
Stock/working solutions: A stock solution of each analyte at
1 mg/ml was prepared in ACN. Working solutions were prepared
in ACN in different concentration levels every alternate day by
appropriately diluting the stock standard solutions and analyzed.
The stock solutions, as well as working standard solutions, were
stored under refrigeration.
Sample Extraction
QuEChERS for tissue: 10 g (± 0.01) of tissue sample cut
into fine pieces was weighed in the 50 ml centrifuge tube and
spiked with known spiking solution followed by addition of
10 ml of acidified ACN (1% acetic acid/ACN). If the tissue
contains sufficient water quantity to achieve partition after
addition of extraction salts, then the addition of water was not
necessary during this step. However, if the tissue is dry, then
2 ml of water was added to achieve the desired consistency.
The tightly capped tube was sonicated for 5 min, and then
Agilent QuEChERS SampliQ extraction salts were added to
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 Deshpande and Srivastava: Pesticide residues in post-mortem samples

the tube. Again, after shaking vigorously for another two min, Specificity: The specificity of the method as demonstrated by
the tube was centrifuged for 3 min at 4000 rpm to get a clear analyzing 15 blank extracts from tissue and blood samples did
upper layer [Figure 1]. 6 ml of the supernatant was transferred not show any interfering peaks [Figure 3b] at the retention time
to the Agilent SampliQ dispersive SPE 15 ml tube and shaken (Rt) of the analytes. The other system suitability parameters
vigorously for 2-3 min and centrifuged for another 3 min to get evaluated by the optimized method by adding pesticides at
a clear supernatant for analysis. the upper limit of calibration range to extracts obtained from
pesticide free tissue and blood established the absence of any
QuEChERS for blood: 0.5 ml of blood was diluted three-fold endogenous interference.
with distilled water in a tube containing 0.5 g pre-packed
extraction preparation (Agilent SampliQ) and a stainless steel Linearity: The calibration plots obtained by plotting the
bead. After spiking with a known spiking solution and adding peak area versus analyte concentration for all the pesticides
2 ml acidified ACN (1% acetic acid/ACN), the tube was shaken
vigorously for a minute and then centrifuged at 4000 rpm
for 5 min. The supernatant (1 ml) obtained [Figure 2] after
centrifugation was then transferred to a 2 ml centrifuge tube
containing SPE sorbent (SampliQ), shaken vigorously and
centrifuged at 4000 rpm for 1 min to get a clear supernatant
for analysis.

Equipment

Liquid chromatography was performed with a binary-solvent

delivery module liquid chromatograph model LC-10ATvp,
(Shimadzu Corporation, Kyoto, Japan) equipped with UV-visible
detector model SPD-10Avp, and column oven model CTO-20A
Prominence. Mobile phase for isocratic elution consisted of Figure 1: Flow chart of QuEChERS extraction method for tissue
ACN: water 60:40 (v/v) mixed together with a binary-solvent
delivery module, model LC-10ATvp. Chromatographic
separation was performed on a Phenomenex C18 endcapped
column (250 × 4.6 mm i.d., particle size 5 μm), (Phenomenex,
USA). Manual sample injector model 7725i, (Rheodyne, Co.,
Berkeley, CA, USA) having a 20 μL sample loop was used.
Winchrom-Ex software module was used for data acquisition
and processing.

RESULTS

The optimized method with a flow of 1.5 ml/min for sample Figure 2: Flow chart of QuEChERS extraction method for blood
20 μL efficiently resolves the six pesticides within 20 min.
A liquid chromatogram in Figure 3a clearly indicates that the
peaks are distinguished from the baseline as narrow, sharp, well
resolved, and non-tailed. The other optimization parameters
considered were peak area, separation efficiency and peak
asymmetry as presented in Table 1.

Table 1: System suitability parameters and separation

characteristics
Pesticide Retention No. of Retention Asymmetry
time±SDa theoretical factor (k) ratio (T) a
tR (min) plates/
25 cm, (N)
Propoxur 3.36±0.01 8211 2.35 1.0
Malathion 7.47±0.02 10709 6.43 0.84
Quinalfos 10.20±0.02 11615 9.15 0.79
Profenofos 16.14±0.02 12124 15.06 0.77 b
Endosulfan β 20.16±0.04 15292 19.05 0.82
Figure 3: (a) Typical liquid chromatogram of the pesticides. The
Endosulfan α 26.21±0.04 13263 24.13 0.81
elution order and peak numbering is as follows: 1. Propoxur, 2.
Chlorpyrifos 27.96±0.04 12829 26.88 0.84
Malathion, 3. Quinalfos, 4. Profenofos, 5. Endosulfan β, 6 Endosulfan α,
a
Represents the standard deviation for the retention time (n=10) 7. Chlorpyrifos, (b) A representative of blank tissue/blood extract

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Deshpande and Srivastava: Pesticide residues in post-mortem samples

show good linearity with correlation coefficients (r) ranging
from 0.9996 to 0.9999. The concentration range used for
tissue varied from 0.5 to 50 μg/g while that for blood varied
from 0.5 to 25 μg/ml. The experimental LOD and limits of
quantitation (LOQ) are based on the standard deviation of the
response and slope. As summarized in Tables 2 and 3, the LOD
ranges from 0.37 to 0.70 μg/g in tissue and 0.14 to 0.35 μg/ml
in blood. The limit of quantitation in tissue and blood ranges
from 1.16 to 2.50 μg/g and 0.44 to 1.50 μg/ml, respectively. The
precision determined as relative standard deviation (RSD) in
terms of repeatability (intra-day) and reproducibility (inter-
day) at three fortification levels for tissues [Table 2] and blood
[Table 3] were satisfactory with the intra- and inter-day RSD
values ≤ 15% and 10%, respectively. The recoveries determined
at two different concentrations were ≥85% for tissue and ≥90%
for blood, which proves the accuracy of the method [Tables 2
and 3].
DISCUSSION
Since the post-mortem tissues are preserved in saturated
salt solution, the sample extraction was based on the AOAC
2007.01 version of the quick, easy, cheap, effective, rugged,
and safe (QuECHERS) method as it does not incorporate
sodium chloride in the initial extraction step, excess of which
could prohibit partitioning and separation of the ACN layer.
As described in the experimental section, the method involves
two steps: Extraction and dispersive SPE (d-SPE). In the first
step, ACN is added to the sample, followed by salting out of
water from the sample by addition of the salts. ACN facilitates
protein precipitation while liquid-liquid partitioning gets
induced by salting out with sodium acetate and magnesium
sulfate thereby making the process non-clumpy and non-
sticky. Pesticides are known to accumulate in fatty tissues of
living organisms including humans. In the second step, d-SPE
minimizes the matrix effects with a combination of PSA, C 18
(octadecylsilane), and anhydrous MgSO4. The amine removes
fatty acids, sugars, and some ionic lipids, whereas C18 effectively
removes the fats and lipids while MgSO4 remove the remaining
water in the extracts.
To optimize the operating conditions, systematic investigation
of the influence of various parameters, viz., nature of bonded
stationary phase, composition, and flow rate of the mobile
phase was studied. Initial scanning of UV spectra for individual
pesticides facilitated selection of detector wavelength at
230 nm. RP C18 columns are commonly available in routine
analytical laboratories and hence chosen for the present study.
The mobile phase was degassed in an ultrasonic bath and filtered
through 0.45 μm membrane (Millipore Corporation, USA)
before use. The method was kept simple by choosing isocratic
elution with varying proportion of ACN and water. To study the
effect of ACN on the retention behavior for all the pesticides
curves corresponding to the Rt versus ACN in the mobile phase
were constructed. The curves clearly indicated that the retention
factor of all the pesticides decrease with increase in ACN in
the mobile phase. Chlorpyrifos and endosulfan-α, overlap at
67% ACN as evident from Figure 4, but further reduction of

Table 2: Performance characteristics of the HPLC-UV method for determination of pesticides in tissue samples
Pesticide LOD at 3.3 σ/S LOQ at 10 σ/S Linearity Correlation Precision at different concentration % Recovery at different
(μg/g) (μg/g) (μg/g) coefficient (r) levels (RSD) (μg/g) spiking levels (μg/g)±SD
Intra-day Rp Inter-day Rc (n=5)
2 10 25 2 10 25 2 25
Propoxur 0.45 1.37 1.37-50 0.9996 13.6 8.4 9.7 14.1 12.1 11.5 82±8 84±4
Malathion 0.51 1.50 1.50-50 0.9997 11.4 6.4 8.3 10.9 12.4 8.3 84±4 87±5
Quinalfos 0.37 1.10 1.10-50 0.9997 11.8 9.2 10.1 13.4 8.5 8.9 86±5 93±3
Profenofos 0.38 1.16 1.16-50 0.9998 10.1 9.8 7.5 12.6 6.2 10.1 85±4 98±3
Endosulfan β 0.70 2.10 2.10-50 0.9996 10.3 11.5 10.2 9.3 7.2 10.5 83±2 90±1
Endosulfan α 0.50 1.60 1.60-50 0.9996 12.5 7.6 6.3 14.8 5.4 4.3 87±2 91±3
Chlorpyrifos 0.70 2.00 2.00-50 0.9998 8.5 3.8 3.3 12.3 7.1 5.3 82±3 98±1
Rp: Repeatability (n=5), Rc: Reproducibility (n=5), HPLC-UV: High-performance liquid chromatography-ultraviolet, LOQ: Limits of quantitation,
LOD: Limits of detection, SD: Standard deviation, RSD: Relative standard deviation

Table 3: Performance characteristics of the HPLC-UV method for determination of pesticides in blood
Pesticide LOD at 3.3 σ/S LOQ at 10 σ/S Linearity Correlation Precision at different concentration % Recovery at different
(μg/ml) (μg/ml) (μg/ml) coefficient (r) levels (RSD) (μg/ml) spiking levels (μg/ml)±SD
Intra-day Rp Inter-day Rc (n=5)
2 10 25 2 10 25 2 25
Propoxur 0.27 0.84 0.84-25 0.9997 6.6 5.1 5.7 8.7 4.2 6.7 92±5 94±3
Malathion 0.19 0.50 0.50-25 0.9998 4.4 5.4 2.7 2.3 1.1 1.2 91±2 96±1
Quinalfos 0.16 0.47 0.47-25 0.9997 8.4 6.3 2.5 4.6 5.3 3.4 94±2 93±1
Profenofos 0.14 0.44 0.44-25 0.9998 7.1 3.6 1.1 5.2 4.1 1.1 91±5 92±2
Endosulfan β 0.52 1.50 1.50-25 0.9996 8.7 5.6 7.3 6.2 2.3 1.7 92±3 95±1
Endosulfan α 0.48 1.01 1.01-25 0.9996 9.4 9.5 6.2 4.8 5.5 3.3 91±6 92±4
Chlorpyrifos 0.70 2.00 2.00-50 0.9998 5.1 3.8 3.3 4.9 4.1 2.3 90±4 98±1
Rp: Repeatability (n=5), Rc: Reproducibility (n=5), LOQ: Limits of quantitation, LOD: Limits of detection, SD: Standard deviation, RSD: Relative
standard deviation, HPLC-UV: High-performance liquid chromatography-ultraviolet

4 Eur J Forensic Sci ● Jul-Sep 2016 ● Vol 3 ● Issue 3


Figure 4: Effect of acetonitrile on retention factor of pesticides
ACN to 60% in the mobile phase separates them efficiently
(resolution > 1.0). The identity of all analyte peaks in samples
was confirmed by comparing their Rts with the Rts of reference
standards under similar HPLC conditions.
The method was evaluated in terms of selectivity, limit of
detection (LOD), linear range of calibration, precision, and
accuracy as per the important considerations mentioned by
Peters et al. (2007) in validation of new methods to be used for
forensic or clinical toxicology. The specificity of the method
which establishes the fact that there is no interfering signal with
that of the analytes was achieved by analyzing 15 different blank
tissue/blood samples and standards spiked in them. The other
system suitability parameters in terms of various retention and
separation characteristics, such as Rt, column efficiency (N),
retention factor (k), peak tailing or asymmetry ratio (T), and
resolution (Rs), were also evaluated to show the absence of
any endogenous interference. The lowest concentration of
the analyte that can be detected is the LOD and the LOQ
that determine the sensitivity of the method are based on the
standard deviation (σ) of the response and the slope (S), where
LOD is 3.3 σ/S and LOQ is 10 σ/S.
Precision was determined in terms of RSD for the repeatability
and reproducibility of the method. Repeatability was assessed by
running five tissue/blood extracts spiked at three concentration
levels (2, 10, and 25 ug/g) while reproducibility was evaluated
by running five replicate samples that were spiked at same
concentrations (2 and 25 ug/g) and analyzed on five different
days. The recovery efficiencies of the spiked samples at the lower
and higher concentrations by the optimized method evaluated
the accuracy of the method. To achieve this, two sets of samples
were prepared: Set 1 was prepared in a blank matrix extract and
spiked after extraction, whereas Set 2 was prepared by spiking
before extraction. Recovery was calculated by relating the areas
of the extracts before and after extraction, as follows:
Recovery % = set 2area/set 1area × 100
The matrix-matched calibration method demonstrates an
effective method of calibration, wherein a series of standard
solutions including one at LOQ level were prepared in blank
extracts of tissue and blood samples and the correlation
Eur J Forensic Sci ● Jul-Sep 2016 ● Vol 3 ● Issue 3
Deshpande and Srivastava: Pesticide residues in post-mortem samples
coefficients (r) of the linear regression curves obtained for each
analyte established the linearity.
The method is applicable only for the quantification of these
pesticides after screening and reliable identification by thin layer
chromatographic technique using target specific chromogenic
spray reagents or any other hyphenated technique.
CONCLUSIONS
The analyst can face many problems during the performance
of any method. In forensic toxicology accurate quantification,
especially in post-mortem tissues is always a subject of debate.
Conventionally, the method of standard addition is preferred
in forensic analyses of post-mortem samples in which the
calibration and quantification are performed in the sample
matrix of the case in question. This can be tedious and time
consuming for routine analysis if the number of analyzes is
sufficiently high. The present validation study has confirmed
that if the samples are properly preserved, matrix-matched
calibration using matrix samples from several post-mortem
samples can be applied for routine analysis of these pesticides
in both blood and tissues with a good precision and accuracy.
QuEChERS sample preparation reduces potential cross-
contamination of samples thereby facilitating rapid and
efficient analyzes of a large number of samples with an ordinary
chromatographic instrumentation. However, the method cannot
be applied to tissues that are decomposed or putrefied since the
composition of such specimens varies considerably.
ACKNOWLEDGMENTS
The authors are thankful to Dr.M.K.Malve, Director and
Shri.S.M.Bakre, Assistant Director, Directorate of Forensic
Science Laboratories, Mumbai, for their support in providing
tissue and blood samples.
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Source of Support: Nil, Conflict of Interest: None declared.
● Jul-Sep 2016 ● Vol 3 ● Issue 3